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Scooby-Doo, Shaggy, and Scrappy-Doo are on their way to a Miss Grimwood's Finishing School for Girls, where they have been hired as gym teachers. Once there, however, they find that it is actually a school for girl ghouls. The pupils include Sibella, the daughter of Count Dracula; Elsa Frankenteen, the daughter of Frankenstein's monster, Winnie, the daughter of The Wolfman; Phantasma (usually called Phanty for short), the daughter of a phantom, possibly The Phantom of the Opera; and Tanis (which is a parody of an Egyptian city), the daughter of The Mummy - all parodies/tributes to the Universal Monsters of the 1930s-40s. Other residents of the school are a floating white hand; an octopus, who is the school's butler; Legs, a spider that helps with the upcoming volleyball match; Miss Grimwood, the headmistress; and her pet dragon Matches (who dislikes Scooby at first, but later becomes friends, and has a strong friendship with Scrappy). Shaggy and Scooby wanted no part of this at first, but eventually they agreed to stay as teachers.

The following morning began with the class and the new teachers taking ballet lessons. Gym class soon starts, with the intent to train the girls for their upcoming volleyball match against the boys of the neighboring Calloway Military Academy. Soon it was time to start the match. For many of the serves it was a seesaw battle. However, thanks to a device that the boys put in the volleyball allowing the boys to win, they started to pull away. But because of an accidental squirt of ketchup, the boys lose the remote, Scooby accidentally swallows the remote and it allows the girls to win instead.

The girls' fathers come for Open House, but as they leave, they warn Shaggy and Scooby not to let any harm come to their daughters or they'll be in big trouble. As an additional plot twist, Revolta, the self-styled Witch of the Web, and her minion, the Grim Creeper, plan to kidnap the girls and make them her slaves, by hypnotizing Shaggy into taking the girls on a field trip to the Baron Bog. That same day the Calloway Cadets are at the bog. With the help of Revolta's spider bats, Revolta and the Grim Creeper capture the girls and Revolta makes a potion that will make them evil forever at the stroke of midnight. Scooby, Scrappy, Shaggy, and Matches, along with the Calloway cadets manage save the girls; and Revolta's plan is foiled.

Despite being well liked by all their students, Shaggy and Scooby have enough of this and run away when monsters such as an alien, the Creature from the Black Lagoon, and Godzilla enroll their daughters at the school for the following year screaming "Like, meet us in St. Louis". As they leave, they see the girls and Matches wave them goodbye. Shaggy, Scooby, and Scrappy then give them a last werewolf howl before driving off into the night.

Miss Grimwood, the middle-aged headmistress of the school. She wears a red cape over a pink dress, a red headband, red shoes, and has short black hair. She has a pet dragon named Matches.

Matches, a small, light-green dragon who serves as Miss Grimwoods's pet guard dog. He often gets duties like heating food in the kitchen or warming up items with his fire breath in which his body will turn a dark red color. However he will not be able to breathe fire if his body is wet. Matches often shows a bad temper. At first, he shows a strong dislike towards Scooby—a recurring gag throughout the film is that Scooby would makes Matches angry and gets his tail set on fire as punishment. Matches later warms up to Scooby after the dog saves him from the Grim Creeper and helps rescue the girls from Revolta.

Sibella, Dracula's pre-teen daughter. She has green eyes, light purple skin, purple hair, red shoes, and a dark purple dress with a red belt. Sibella can turn into a bat at will, which she uses for important reasons such as rescuing or looking for people, or for simple things such as delivering a pizza. Unlike traditional vampires like her father, she is immune to sunlight and can go out during the day. She is also shown to be the most strongly willed of the five Grimwood girls—she is the last to undergo Revolta's hypnosisspell and tries to fight back. She often uses phrases with vampire-related puns such as "Fang-tastic" and "You bat" (you bet). She is talented at making bat-robes (bathrobes) and dragon fire-proof robes as seen when she gives her father and Matches each one. She is soft and down-to-earth, and is considered the one Shaggy and Scooby took longest to warm up to.

Elsa Frankenteen, Frankenstein's monster's daughter. Elsa has platform-like brown sandals and a green dress with a light green T-shirt underneath. She can endure electric shocks and her black hair is standing up above her head thus resembling the Monster's bride from the Bride of Frankenstein. She presents her father with a portable shockman that has a rechargeable battery-pack that lasts for weeks, showing that her father can also endure electric shocks. She is the tallest and the strongest of the girls. She has a deep masculine voice. She is based on the actress, Elsa Lanchester, who portrayed the Bride of Frankenstein.

Winnie, the Wolfman's daughter; quite often Winnie would howl before making a statement and does not turn back into a human during daylight hours. She wears a blue dress, a light blue bow-tie and has light brown fur and orange, curly hair. Winnie knows how to make a good juicer for making bitter lemonade as she made one for her father in the film. She talks the most out of all the girls. She is competitive and loud.

Phantasma, a phantom's daughter, presumably The Phantom. Everyone calls her "Phanty" for short. With a shrill, high laugh, she has short white hair with a blue streak and pale blue skin. She wears a blue dress and white boots. Being intangible, she can float through walls and doors. She has a good sense of humor and loves music, and is musically gifted. Phanty's gift to her father was her latest composition which was called "Duet For Three Hands and Six Tentacles".

Tanis - The Mummy's daughter, Tanis is the smallest and youngest of the girls. She has big, blue eyes and a bandage ponytail embellished with a dark pink bow. She usually gets tangled in her bandages and the others have to untangle her and re-wrap her. She calls her daddy "Mummy-Daddy." Tanis has an Egyptian trophy case in which to store trophies, but she often gets worried when it looks like they are going to lose a game. Tanis gave her father a fright-light to brighten the darkest mummy case for a present. She is a shy sweetheart, and is the most nervous out of the group of girls. Scrappy warms up to her immediately, mostly because of her innocence. She often sucks on her thumb to show her innocence.

In the final scene of the film, the daughters of an alien, the Creature from the Black Lagoon, and Godzilla come to attend but Shaggy, Scooby and Scrappy drive off before we get to know them.

Calloway cadets

A group of boys of Colonel Calloway's Military School, located next to Grimwood. They often use the terms "affirmative" or "negative" when agreeing or disagreeing with particular happenings.

Colonel Calloway, the head of Calloway Military School. Fair yet very stern, Calloway seems to have a reciprocated attraction to Miss Grimwood.

Tug Roper, the leader of the cadets. He often tries to maintain his dignity and even resorts to cheating during the volleyball game against the Grimwood Girls, but he and his team still lose. He does what he can to impress the colonel, but often disappoints him with his clumsiness. He is the male equivalent of Sibella.

Miguel, the inventor of the group, most of the time his inventions are messed up by the others. He is the male equivalent of Phantasma.

Jamal Williams, the tallest and most skilled of the cadets, despite not being the leader, Col. Calloway seems to admire him more than Roper; he always answers to every positive sentence with "affirmative". He is the male equivalent of Elsa.

Grunt, the largest member of the cadets; despite being overweight, he is very athletic and very strong. He is the male equivalent of Winnie.

Baxter, the smallest member of the group. He seems to be the most obedient of the cadets; following the colonel's every word and acknowledging him at almost every time. He is the male equivalent of Tanis.

Revolta- The Witch of the Web. She plans to make the students at the ghoul school part of her evil squad permanently. Her main magic is mind control via spider bats who put web-like hypnosis headphones on the victim and Revolta issues commands through that headphone which hypnotizes the victims. She is also a good potions maker and can brew potions that make others evil permanently. She is presumed destroyed at the film's conclusion.

Grim Creeper- Revolta's assistant. He looks like a green, overgrown potato with long vines for limbs and one eye in the middle of his head. He unsuccessfully tried to get the wand from Revolta when Matches came near him and was presumably destroyed alongside Revolta.

Mirror Monster- A Monster who lives in one of Revolta's mirrors and can change into anyone that looks into it. In the movie, the monster traps Shaggy inside the mirror and replaces him. He chases after Scooby, who eventually fools him into thinking he is one of Revolta's slaves. In the end, Matches burns his backside, and he jumps back into the mirror, setting Shaggy free.